Analogues of angiotensin II

ABSTRACT

Novel hepta- and octapeptides of the formula:   WHEREIN R is hydrogen, succinyl, L-aspartyl, L-sarcosyl, Lseryl, succinamyl, or D- or L-asparaginyl and R1 is an L-alanine, L- or D-leucine, glycine or L-isoleucine residue upon intravenous infusion to animals inhibit blood pressure response to angiotensin amide.

United States Patent [191 Sipos et al.

[451 May27, 1975 ANALOGUES OF ANGIOTENSIN II [75] Inventors: Frank Sipos, Norwich; Donald T. Pals, Oxford; George S. Denning, Norwich, all of NY.

[73] Assignee: Morton-Norwich Products, Inc.,

Norwich, NY.

[22] Filed: June 4, 1970 [21] Appl. No.: 43,595

Related US. Application Data [63] Continuationin-part of Ser. No. 17,920, March 9,

1970, abandoned.

OTHER PUBLICATIONS Bumpus et al., Biochim. Biophys. Acta 46, 38-44 (1961).

Park et 2]., Biochemistry (Wash) 6, 3458-3464 (1967).

Bumpus et a1., Peptides:Chemistry and Biochemistry, Weinstein et 211., eds. Marcel Dekker, Inc., New York (1970) pp. 127-150 Effective date Aug. 1968.

Schroder et al., The Peptides, Vol. 11, Academic Press, New York (1966), pp. 47-62.

Khairallah et a1., .1. Med. Chem. 13, 181-184 (1970).

Khosla et al., Biochemistry (Wash) 2, 3417-3421 (1968).

Primary Examiner-Elbert L. Roberts Attorney, Agent, or FirmAnthony J. Franze [57] ABSTRACT Novel heptaand octapeptides of the formula:

wherein R is hydrogen, s'uccinyl, L-aspartyl, L- sarcosyl, L-seryl, succinamyl, or D- or L-asparaginyl and R is an L-alanine, L- or D-leucine, glycine or L- isoleucine residue upon intravenous infusion to animals inhibit blood pressure response to angiotensin amide.

14 Claims, N0 Drawings ANALOGUES F ANGIOTENSIN ll tive to penetrate the resin. Then 8.5 mMol. of This application is a continuation-in-part of our co- N,N' dic cl hexylcarbodiimide (DCC) in ml. methpending application Ser. NO. 17.920, filed ar. 9, 1970 ylenechloride was added and the coupling was allowed and now abandoned. to proceed for 12 hours with rocking. Thereafter the This invention relates to polypeptides. More particus resin was washed with three portions each of methyllarly it is concerned with heptaand octapeptides of the enechloride, absolute ethanol and acetic acid and so formula: was prepared for the next deprotection step with HCl H 2 N-C NH N I l N llw H \v/ci i H C\ CH (615 B ca CH tr: CH

I l 2 l R-NH-C E1-C ONH CPI-C ONIrL-CILC ONE-C H- C ONH- CILC ON C H-C 0- R wherein R is hydrogen, succinyl, L-aspartyl, sarcosyl, in acetic acid as described above. The washing, neutral- L-seryl, succinamyl, or D- or L-asparaginyl and R is an ization and coupling steps were performed by the de- L-alanine, L- or D-leucine, glycine or L-isoleucine resi- 2O scribed method using BOC-His(N""-Bzl)-OH, BOC- due. Val-OH, BOC-Tyr(O-BZl)-OH and BOC-Arg(NO The peptides of this invention possess pharmacolog- OH. Change was made in the coupling steps of the ical activity. They are capable of inhibiting the pressor BOC-His(N""-Bzl)-OH and BOC-Arg(NO )-OH in effect of angiotensin amide upon blood pressure. Thus which cases a dimethylformamide-methylenechloride when administered by intravenous infusion to pithed (2: 1) mixture was used as solvent. The coupling of Asn rats in the very small amount of 20 mcg./kg,/min., the to the deprotected resin-heptapeptide was performed pressor effect of angiotensin amide similarly adminisusing BOC-Asn-ONP (BOC-Asparagine-ptered is inhibited. By virute of this inhibitory property nitrophenylester) in dimethylformamide by rocking the upon angiotensin amide induced blood pressure eleva mixture for 72 hours. tion. the peptides of this invention are valuable agents After the last coupling step the resin-peptide was for counteracting hypertension due to angiotensin amwashed with dimethylformamide, ethanol, acetic acid ide. They are also capable of inhibiting hypertension in and ethanol and dried in vacuo over P 0 The weight acute unilateral renal hypertensive rats upon intraveof the resin-peptide was 7.3 g. nou i f io H-Asn-Arg(NO )-Val-tyr(O-Bzl)-Val-His(N""-Bzl)- The heptaand octapeptides of this invention are PrO-AlH-OHQHBr (B) readily prepared in accordance with known methods The resin-peptide I (7.3 g), prepared as described for preparing peptides. Such methods involve the buildabove, was suspended in 25 ml. dry trifluoroacetic acid ing of a linear chain of amino acids through repetitive and a stream of dry HBr was passed through at a slow amide linkages employing in such sequential alignment rate. After 20 min. the resin was filtered off and treated the necessary protective groups susceptible of ready once more with HBr/CF COOH for 40 min. The filremoval by conventional cleavage methods. The adaptrates were evaporated to dryness in vacuo at 20, the tation of such methods to the peptides of this invention oily residues were precipitated with abs. ether and the is described hereinafter in exemplary fashion by the folproduct was dried in desiccator over potassium hydroxlowing: ide. The overall yield was 1980 mg. (59.8%) of the pro- L-Asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L- tected octapeptidedihydrobromide. histidyl-L-prolyl-L-alanine: H-Asn-Arg-Val-Tyr-Val-His-lPro-Ala-OH (C) BOC-Asn-Arg(NO )-Val-Tyr(O-Bzl)-Val-His(N The protected peptide (B) (1.0 g) was dissolved in 20 Bzl)-Pro-Ala-polymer(A) ml. of an acetic acid-dioxane-water (4:4: 1 v/v) mixture BOC-Ala-reinester (5 g, 0.5 mMol/g) was placed in and hydrogenolysed over Pd/BaSO 10% catalyst (0.5 a rocking type 200 ml Merrifield reaction vessel. The g) for 48 hrs. at atmospheric pressure. After that time resin was swelled in chloroform (analytical grade) by a new portion (0.2 g) of catalyst was added and the hyrocking for 20 min. and thereafter washed with three drogenation was continued for 24 hours. The filtered 50 ml portions of glacial acetic acid. The duration of and diluted solution was lyophilized. The yield was 750 each wash operation was 35 min. The t mg- (m.p- 150l65). (Calcd. for dihydrobromide: butoxycarbonyl(BOC) protecting group was removed 15.32% Br. Found: 13.36% Br.) by IN HCl in anhydrous acetic acid by rocking for 40 min. The resin was successively washed three times The Crude Product was Purlfied y g Permeatlon each with acetic acid, absolute ethanol and with chfomatography on seph'aqex in acetic N,N'-dimethylformamide. The resulting hydrochloride acld- The fractlons comammg the p p p were of alanylresinester was neutralized with a 10% solution comblned and y p The purified Compound of triethylamine in dimethylformamide by rocking for comams only a trace of halogen 10 min. Thereafter th r sin was washed with three TLC: Merck Cellulose F plates, n-butanol-acetic portions each of dimethylformamide, abs. ethanol, acid water (612:3 v/V) solvent System, R! 0.35 chloroform and methylenechloride and the solution of Merck Silica Gel R254 plates with the same SOL 8.5 mMol (three fold excess) of BOC-proline in 40 ml. vent SystemRf o 1 3 of methylenechloride was added. A 20 min. rocking pe- 4 594 0,2, IN Ac OH)[a] 6 l .1 (c

riod was allowed to give time for the amino acid deriva- 0 2 {N OH) Amino Acid Analysis:

Ala: 1.00; Arg: 1.00; Asn: 1.09; His: 0.90; Pro:

1.04: Tyr: 0.75; Val: 1.94.

In similar fashion other peptides of this invention were prepared introducing at the appropriate stage the requisite unit. The peptides thus prepared are set forth in the tables herebelow:

TABLE I TLC on TLC on Merck Silica Gel F-254 Merck Cellulose F plates plates Specific Peptide Rotation butanol-acetic acid-water (6:213 v/v);

ninhydrin detection (Concentration R, R, Formula, mol. weight and solvent) H-asn-Arg-Val-Tyr-Val-His-Pro-Ala-OH [111 8 1.33 0.05 0. l

[C H N, O,,, m.w. 9551] (c 0.22, IN Ac OH) H-Asn-Arg-Val-Tyr-Val-His-Pro-Leu-OH [01],, 70.55 0. l5 0.34

[C., H N,.,O,,, m.w. 997.2] (0 1, IN Ac OH) H-asn-Arg-Val-Tyr-Val-His-Pro-Leu-OH [v1] -87.32 0.10 0.45

[C H N O m.w. 997.2] (c 0, 2, IN Ac OH) Succinyl-Arg-Val-Tyr-Val-His-Pro-Ala-OH [a], m 64.90 0.28 0.50

(Paulys reagent) (Paulys reagent) [C H N 0, m.w. 941.1] (0 1, IN Ac OH) H-Asp-Arg-Val-Tyr-Val-His-Pro-Ala-OH [01],, =l01.62 0.04 0,05

[C H N, O, m.w. 956.1] (c =0, 3, IN Ac OH) H-Arg-Val-Tyr-Val-His-Pro-Ala-OH [04],, -68.31 0.22 0.38

(c 0.3 IN Ac OH) TABLE II TLC on Merck Silica Gel F-254 Plates Specification Rotation (ninhydrin detection) (C ,,H N O m.w. 997.22)

Peptide butanol-acetic butanol-acetic acidacidyridine- (Formula, mol. weight) Concentration, Solvent) water (612:3: v/v) water :2:7:6: v/v) H-Sar-Arg-Val-Tyr-Val-His-Pro-Ala-OH 84.4 1;

0.05 0.30 (C H N OW m.w. 912.11) (c=0.2, IN Ac OH) H-Ser-Arg-Val-Tyr-Val-His-Pro-Ala-OH -69.00;

0.02 0.25 (C.,,H,,,,N,,,O,,, m.w. 928.11) (c=0.2, lN Ac OH) H-Asn-Arg-Val-Tyr-Val-His-Pro-leu-OH 4.40;

0.15 0.50 (C H N O m.w. 997.22) (c=0.25, IN Ac OH) Succinamyl-Arg-Val-Tyr-Val-His-Pro-Ala-OH -83. 14;

0.10 0.20 (C.,;,H N, O11, m.w. 940.12) (c=0.25, IN Ac OH) H-Asn-Arg-Val-Tyr-Val-His-Pro-Gly-OH -54.90;

v 0.10 0.25 (C., H .,N,.,O,,, m.w. 941.11) (c=1.0, 1N Ac OH) H-Asn-Arg-Val-Tyr-Val-His-Pro-Ile-OH -55.33;

(c=0.3, IN Ac OH In the above tables, in accordance with IUPAC What is claimed is: 1. A peptide of the formula:

R-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L- prolyl-R, wherein R is hydrogen, succinyl, L-

aspartyl, sarcosyl, L-seryl, succinamyl, or D- or L- asparaginyl and R, is L-alanine, L or D-leucine,

glycine or L-isoleucine.

2. The compound of claim 1 wherein R is hydrogen and R, is L-alaninc.

3. The compound of claim 1 wherein R is L- asparaginyl and R, is L-alanine.

8. The compound of claim 1 wherein R is succinyl and R, is L-alanine.

9. The compound of claim 1 wherein R is sarcosyl and R, is L-alanine.

10. The compound of claim 1 wherein R is L-seryl and R, is L-alanine.

11. The compound of claim 1 wherein R is L- asparaginyl and R, is D-leucine.

12. The compound of claim 1 wherein R is succinamy] and R, is L-alanine.

13. The compound of claim 1 wherein R is L- asparaginyl and R, is glycine.

14. The compound of claim 1 wherein R is L- asparaginyl and R, is L-isoleucine.

PM 050 UNITED STATES I PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,886,134 Dated y 5 Inventor s Frank Sipos, Dona1d T. Pals, George S. Banning, Jr.

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

I. N aming o1 inventors: George S. Denm'ng should be George S. Denm'ng, J r

2. In the Abstract in the hne beneath the structura1 formu1a the "L-' preceding "sarcosy1 should be omitted.

3. In co1umn 2, 11'ne'34: "tyr' should be --Tyr-- Signed and Scaled this twenty-third D 21) Of September I 9 75 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN .-1!r 'sting Officer ('mnmissiunvr of Iurcnls and Trademarks 

1. A PEPTIDE OF THE FORMULA: R-L-ARGINYL-L-VALYL-L-TYROSYL-L-VALYL-L-HISTIDYL-L-PROLYL-R1 WHEREIN R IS HYDROGEN, SUCCINYL, L-ASPARTYL, SARCOSYL, LSERYL, SUCCINAMYL, OR D- OR L-ASPARAGINYL AND R1 IS LALANINE, L- OR D-LEUCINE, GLYCINE OR L-ISOLEUCINE.
 2. The compound of claim 1 wherein R is hydrOgen and R1 is L-alanine.
 3. The compound of claim 1 wherein R is L-asparaginyl and R1 is L-alanine.
 4. The compound of claim 1 wherein R is D-asparaginyl and R1 is L-alanine.
 5. The compound of claim 1 wherein R is L-asparaginyl and R1 is L-leucine.
 6. The compound of claim 1 wherein R is D-asparaginyl and R1 is L-leucine.
 7. The compound of claim 1 wherein R is L-aspartyl and R1 is L-alanine.
 8. The compound of claim 1 wherein R is succinyl and R1 is L-alanine.
 9. The compound of claim 1 wherein R is sarcosyl and R1 is L-alanine.
 10. The compound of claim 1 wherein R is L-seryl and R1 is L-alanine.
 11. The compound of claim 1 wherein R is L-asparaginyl and R1 is D-leucine.
 12. The compound of claim 1 wherein R is succinamyl and R1 is L-alanine.
 13. The compound of claim 1 wherein R is L-asparaginyl and R1 is glycine.
 14. The compound of claim 1 wherein R is L-asparaginyl and R1 is L-isoleucine. 